Prenatal folic acid supplement/dietary folate and cognitive development in 4-year-old offspring from the Japan Environment and Children’s Study

We evaluated the association between maternal prenatal folic acid supplement use/dietary folate intake and cognitive development in 4-year-old offspring (N = 3445) using data from the Japan Environment and Children’s Study. Cognitive development was evaluated using the Kyoto Scale of Psychological Development 2001. Multiple regression analysis revealed that offspring of mothers who started using folic acid supplements pre-conception had a significantly higher language-social developmental quotient (DQ) (partial regression coefficient 1.981, 95% confidence interval 0.091 to 3.872) than offspring of mothers who did not use such supplements at any time throughout their pregnancy (non-users). Offspring of mothers who started using folic acid supplements within 12 weeks of gestation had a significantly higher cognitive-adaptive (1.489, 0.312 to 2.667) and language-social (1.873, 0.586 to 3.159) DQ than offspring of non-users. Regarding daily dietary folate intake from preconception to early pregnancy, multiple regression analysis revealed that there was no significant association with any DQ area in the 200 to < 400 µg and the ≥ 400 µg groups compared with the < 200 µg group. Maternal prenatal folic acid supplementation starting within 12 weeks of gestation (but not adequate dietary folate intake from preconception to early pregnancy) is positively associated with cognitive development in 4-year-old offspring.

Design and participants. The JECS is a nationwide, prospective, birth cohort study involving 100,000 mother-offspring pairs, started in 2011 9,10 . It is ongoing and is planned to continue until the offspring turn 18. Trained examiners evaluated the cognitive development of approximately 5000 offspring selected for the Sub-Cohort Study of the JECS 11 . The dataset of 4-years-old offspring's test results was provided to us in 2021.
We followed the same method as we did for our previous study in which we used the Kyoto Scale of Psychological Development 2001 (KSPD) on 2-year-old offspring 8 . The main differences were that we used the KSPD data of 4-year-old offspring and evaluated sex differences.
Exposure: maternal folic acid supplement use. The Ministry of Health, Labor and Welfare in Japan recommends the intake of 400 µg/day of supplementary folic acid for pregnant women and women intending to become pregnant 12 . A face-to-face interview was conducted with pregnant women to assess their use of folic acid and other supplements 13,14 . In this study, multivitamin supplements were not considered folic acid supplements, as we did not have data on the contents of each multivitamin supplement.
Participants were classified into four groups, based on the time of initiation of folic acid supplementation: (1) preconception users (started before conception), (2) early post-conception users (within 12 weeks of gestation), (3) late post-conception users (after 12 weeks of gestation), and (4) non-users (non-use of folic acid supplements before conception and during gestation).
Exposure: maternal dietary folate intake. A semi-quantitative food frequency questionnaire (FFQ) was used to estimate participants' dietary folate intake from foods 13 . The FFQ comprises a list of foods with standard portion sizes commonly consumed in Japan 15 . The validity of the FFQ for the estimation of dietary folate intake in Japan has previously been established 15 . The FFQ consisted of 172 food and beverage items and nine frequency categories, ranging from almost nothing to seven or more times per day for food and 10 or more glasses per day for beverages. Thereafter, the intake of 53 nutrients was calculated.
The mother's FFQ was administered during the first and second trimesters of gestation, at a median of 14.6 (interquartile range: 12.0-17.9) weeks of gestation. Participants reported their frequency of food consumption over the previous year.
The FFQ was not designed to estimate folic acid 13,15 . The Ministry of Health, Labor and Welfare in Japan recommends an estimated average requirement for total dietary folate, for example, from natural food sources, as follows: an intake of ≥ 200 µg/day for adult women and ≥ 400 µg/day for pregnant women 12 . Therefore, the study participants were classified into three groups, according to daily dietary folate intake (< 200 µg, 200 µg to < 400 µg, and ≥ 400 µg).
Outcome: psychological development of 4-year-old offspring. The KSPD is a standardized developmental assessment tool for Japanese children, covering cognitive-adaptive and language-social areas 16,17 . These areas correspond to nonverbal and verbal cognitive development, respectively. Scores are combined to form the developmental quotient (DQ; in days), which is calculated by dividing the developmental age (in days) by the chronological age (in days) and multiplying the quotient by 100. To ensure reliability of administration of www.nature.com/scientificreports/ the KSPD, the interviewers were trained and certified by the JECS. Administrative procedures and evaluations were strictly standardized to ensure inter-interviewer reliability.
Statistical analysis and covariables. We compared the mothers' characteristics and their offspring's cognitive developmental data via analysis of variance (ANOVA) and Tukey's range test. Sex differences were also examined. Multiple regression analyses were used to assess the association between maternal prenatal folic acid intake/dietary folate intake and offspring psychological development. First, multiple regression analyses were adjusted for maternal age at delivery, maternal body mass index (kg/m 2 ) before pregnancy, infertility treatment, unexpected pregnancies, parity, marital status, maternal highest level of education, paternal highest level of education, maternal smoking status during pregnancy, paternal smoking status during pregnancy, maternal alcohol consumption during pregnancy, annual household income (× 10 3 yen/year) during pregnancy, pregnancy complications, obstetric labor complications, mode of delivery, maternal neuropsychiatric disorders, and a six-item maternal Kessler Psychological Distress Scale (K6) score ≥ 5 during pregnancy [18][19][20] . Adjustments were also made for the sex of the offspring (not for subgroup analysis), the offspring's birth weight, gestational week of delivery, breastfeeding at 18 months postpartum, family structure, maternal job status after delivery, day care center attendance, multivitamin supplement use, iron preparation use, and trace element use. Dietary intake (measured with the FFQ) included energy content and nutrients, including amino acids, n − 3 unsaturated fatty acids, Fe, Ca, vitamin A, vitamin B 12 , and vitamin C. No multicollinearity was observed in the multiple regression analysis. For reference, the parity and number of the offspring's siblings were confirmed to be multicollinear. The total energy, protein, and Zn contents were also confirmed to be multicollinear.
Second, multiple regression analyses were adjusted for variables selected using a stepwise method. All analyses were significant at a 0.05 probability of significance and were performed using SAS statistical software, version 9.4 (SAS Institute Inc., Cary, NC, USA).

Results
We analyzed the records of 3445 offspring out of the 104,059 records in the dataset (Fig. 1). Table 1 summarizes the participants' characteristics. The maximum dietary folate intake in the ≥ 400 µg/day group was 2956 µg/day.

Folic acid supplements.
The results of the ANOVA and Tukey's range test for maternal folic acid supplement use and the KSPD score of the offspring are summarized in Table 2.
In female offspring, the multiple regression analysis without the stepwise method revealed significantly higher score for cognitive-adaptive DQ was observed among early post-conception users (B: 1.670, 95% CI 0.104 to 3.236, β: 0.055, P = 0.04) than among non-users ( Table 3). The multiple regression analysis with the stepwise method that revealed significantly higher score for cognitive-adaptive DQ was observed among early postconception users (B: 1.673, 95% CI 0.211 to 3.135, β: 0.055, P = 0.02) than among non-users (Table 3).   Table 4. Overall, the multiple regression analysis without the stepwise method revealed no significant association with any DQ score in the 200 µg to < 400 µg group or the ≥ 400 µg group compared with the < 200 µg group ( Table 5). The multiple regression analysis with the stepwise method revealed no significant association with any DQ score in the 200 µg to < 400 µg group or the ≥ 400 µg group compared with the < 200 µg group (Table 5).
In male and female offspring, the multiple regression analysis without the stepwise method revealed no significant associations with any DQ score in the 200 µg to < 400 µg group and the ≥ 400 µg group compared with the < 200 µg group ( Table 5). The multiple regression analysis with the stepwise method revealed no significant www.nature.com/scientificreports/ associations with any DQ score in the 200 µg to < 400 µg group and the ≥ 400 µg group compared with the < 200 µg group (Table 5).

Discussion
Our study demonstrated that the offspring of mothers who started prenatal folic acid supplement use within 12 weeks of gestation exhibited better nonverbal and verbal cognitive development at age 4 than did those with mothers who did not use folic acid supplements. However, offspring of mothers with an adequate daily dietary folate intake from preconception to early pregnancy did not exhibit better nonverbal or verbal cognitive development at age 4 than did offspring of mothers with inadequate folate intake in that period. These results are inconsistent with those of our previous study on 2-year-old offspring 8 . First, our previous study of 2-year-old offspring 8 demonstrated that the offspring of mothers who took ≥ 200 µg folate per day from preconception to early pregnancy had a significantly higher DQ in the language-social area than did those in the < 200 µg group. Moreover, the DQ was higher in the ≥ 400 µg group than in the 200 to < 400 µg group. However, this study revealed that the beneficial association was no longer present in 4-year-olds. This suggests that the benefit of maternal dietary folate intake during early pregnancy on the offspring's verbal cognitive development may last for up to approximately 2 years, and that postnatal environment factors may offset the difference between groups by the time the offspring are 4 years of age.
Second, regarding maternal prenatal folic acid supplementation, previous studies 8 and the current study were limited by the lack of detail on the amount of folic acid in the supplements used and the frequency of use. Our previous study of 2-year-old offspring revealed no significant association between starting prenatal folic acid supplement use within 12 weeks of gestation and verbal or nonverbal cognitive development. However, to our surprise, such associations were observed for 4-year-old offspring in this study. In Japan, only approximately 30% of pregnant women seem to start using folic acid supplements before conception or within 12 weeks of gestation 14 . Therefore, pregnant women who use folic acid supplements may be more conscious of their future offspring's health than women who do not [21][22][23] . We hypothesize that mothers who use folic acid supplements in early pregnancy for their offspring's health will exhibit enthusiastic parenting behavior after delivery. The effects of enthusiastic postpartum parenting behavior may become apparent when the offspring reach the age of 4. In www.nature.com/scientificreports/ support of this hypothesis, a cohort study in the Netherlands 24 revealed that there was no association between plasma folate concentrations in pregnant women and autistic traits in their offspring, but that prenatal folic acid use was associated with fewer autistic traits in the offspring at age 3. They suggested that prenatal folic acid supplement use, a marker of good health literacy, is associated with many health-conscious behaviors that decrease the background risk of autistic traits in offspring. To substantiate our hypothesis, the next task would be to analyze the relationship between maternal prenatal folic acid supplement use and postpartum parenting behavior.
In this study of 4-year-old offspring, we also explored sex differences in the effect of folic acid supplementation/dietary folate intake. Male offspring of mothers who started using folic acid supplements before conception or within 12 weeks of gestation exhibited better verbal cognitive development than those of mothers who did not use such supplements. However, no significant association was observed with nonverbal cognitive development. Female offspring of mothers who started using folic acid supplements within 12 weeks of gestation exhibited better nonverbal cognitive development than those of mothers who did not use such supplements. However, no significant association was observed with verbal cognitive development. In animal studies, sex differences have been demonstrated in maternal folic acid loading and behavior in offspring 25,26 ; however, few reports on such sex differences have been made for human studies. The reason for the observed sex difference in human offspring is unknown, and further investigation is required.
For reference, we compared our results to those of previous cohort studies on prenatal folic acid/dietary folate intake and cognitive development in offspring from 3 to 6 years of age 3 . Maternal supplement use of more than 600 µg/day and folate intake from food in early pregnancy were positively associated with receptive language development in 3-year-old offspring in a US cohort study 27 . Maternal folic acid supplement use from the eighth week of pregnancy was associated with a reduced risk of severe language delay in 3-year-old offspring in a Norwegian cohort study 28 . In a US cohort study, the use of periconceptional folic acid supplements was not associated with language development in 3-year-old offspring 29 . In a Spanish cohort study, a positive association between maternal use of folic acid supplements at the end of the first trimester and social competence, verbal skills, and verbal-executive skills was observed in 4-year-old offspring. However, no differences in perceptive performance or memory were observed in that study 30 . In a European, multicenter, randomized controlled trial, the maternal use of 400 μg/day folic acid supplement from the 20th week of pregnancy until delivery had no significant effect on the cognitive function of 6.5-year-old offspring 31 . Moreover, a study in the US revealed that the folate nutritional status of mothers in the latter half of pregnancy, assessed via plasma and erythrocyte folate concentrations, had no impact on the cognitive development of 5-year-old offspring 32 .
This section discusses the overall importance of folate/folic acid in pregnancy. Because the fetus receives folate from the mother through the placenta, pregnant women's folate/folic acid intake must be adequate. Folate deficiency in pregnant women can cause megaloblastic anemia 1 . Low folate status in pregnant women increases the risk of preterm delivery, low birth weight, fetal growth retardation, congenital heart disease, and structural malformations such as oral clefts 1 . It has also been suggested that maternal folate deficiency may result in neurodevelopmental disorders such as autism spectrum disorders and schizophrenia in their offspring 1 . It is also well known that supplementation with folic acid, a synthetic form of folate, reduces the prevalence of folate deficiency during pregnancy and that folic acid supplementation during gestation reduces the risk of neural tube defects (NTD) in the fetus [3][4][5] .
Besides folate/folic acid, other nutrients, such as protein, zinc, iron, vitamins, and long-chain polyunsaturated fatty acid, are also important for offspring's neurodevelopment [33][34][35] . The nurturing environment after birth is also important. Therefore, factors other than those we have included as confounding factors in this study may also likely play a role in children's neurodevelopment. Further comprehensive evaluations that include folate/ folic acid and these factors are needed.
This study had some limitations. The first limitation was the retrospective collection of information for maternal supplement use, which in the case of the preconception period was at least 10-16 weeks before the interviews; this may not have been very accurate. Second was the lack of detailed information on the use of folic acid supplements and whether the supplements used by all of the study participants contained the same amount of folic acid. In Japan, folic acid supplements are manufactured by various companies, but pregnant women and women planning to conceive are recommended to supplement their diet with 400 μg/day of folic acid, not exceeding 1000 μg/day 12 . Thus, although not all pregnant women necessarily received the same dose of folic acid, each likely consumed at least 400 μg/day.Third, there was no accurate information on how long women took folic acid supplements during preconception or pregnancy. Fourth, the fact that dietary folate intake was self-reported via the FFQ. Fifth, there was no information on reliable biochemical indicators of folate status, such as red blood cell folate concentration, in the JECS study.
However, the study's strength was in the objective investigation of the offspring's cognitive development by trained interviewers.
In conclusion, our study demonstrated that maternal prenatal folic acid supplement use starting within 12 weeks of gestation was associated with higher verbal and nonverbal cognitive development in 4-year-old offspring than not using such supplements. However, there were sex differences in this association. Offspring of mothers with an adequate daily dietary folate intake from preconception to early pregnancy were not at an advantage in terms of verbal and nonverbal cognitive development at 4 years of age.

Data availability
Data are unsuitable for public deposition due to ethical restrictions and legal framework of Japan. It is prohibited by the Act on the Protection of Personal Information ( www.nature.com/scientificreports/ access to data should be sent to: jecs-en@nies.go.jp. Te person responsible for handling enquiries sent to this e-mail address is Dr. Shoji F. Nakayama, JECS Programme Office, National Institute for Environmental Studies.